Tumor cell metastasis--a process whereby cells from a primary tumor migrate from the tissue of origin in the circulatory or lymphatic systems to establish secondary tumors in other tissues--involves the ability of cells to bind to and invade basement membrane, the thin extracellular matrices that surround epithelial tissues, nerves, fat cells, and smooth, striated, and cardiac muscle. Since basement membrane surrounds tissues and blood vessels, this barrier must be crossed several times during metastasis. First, though, specific cell-surface receptors must bind to various glycoproteins that mediate the attachment of the metastasizing cells to basement membrane. Three common attachment proteins are laminin, fibronectin, and chondronectin.
Laminin is particularly interesting because of its localization to basement membrane and because of the preference shown by various carcinomas (for example, colon and breast) to use laminin for cell attachment (Liotta (1986) Cancer Res 46:1-7; Terranova, et al, (1983) Proc Natl Acad Sci USA 80:444-448; Terranova, et al, (1982) Cancer Res 42:2265-2269; and Vlodavsky and Gospodarowicz (1981) Nature 289:304-306). Treating certain metastatic cells with an antibody to laminin decreases their ability to interact with basement membrane and reduces the number of metastases produced when the cells are injected into mice (Terranova, et al, (1982) supra).
The function of laminin in attaching tumor cells to basement membrane can be demonstrated by simple in vitro assays that measure either the binding of .sup.125 I-labeled laminin to tumor cells (Rao, et al, (1983) Biochem Biophys Res Comm 111(3):804-808) or the laminin-mediated binding of tumor cells to basement membrane collagen (Terranova, et al, (1982) supra). These assays are conducted using tumor cells from mice or humans. A large fragment (M.sub.r =300,000) of the laminin molecule can be isolated by proteolytic digestion with pepsin (P.sub.1 fragment). The P.sub.1 fragment contains the cell receptor binding site for laminin but has no collagen-binding activity (Charonis, et al, (1985) J Cell Biol 100:1848-1853). P.sub.1 inhibits the binding of tumor cells to basement membrane (Barsky, et al, (1984) J Clin Invest 74:843-848) and to basement membrane collagen (Terranova, et al, (1983) supra). Pretreating mouse tumor cells with P.sub.1 greatly reduces the metastasis of these cells in animals, as demonstrated by the reduction in lung metastases in mice following intravenous (iv) administration of the treated cells (Barsky, et al, (1984) supra). Thus, the inhibition of in vitro laminin binding reflects in vivo antimetastatic activity.
The human laminin B.sub.1 chain has been cloned and sequenced at the nucleotide level (Pikkaraimen, et al, (1989) J Biol Chem 262:10454-10462); however its large size (1765 amino acids minus its signal peptide) prevents its use as a therapeutic agent. Synthetic peptides have been prepared to sequences from various regions of the mouse B.sub.1 chain (Sasaki, et al, (1987) Proc Natl Acad Sci USA 84:935-939), and specific antibodies have also been prepared against some of these peptides. The antibody to a peptide from domain III, a cysteine-rich region of homologous repeats, inhibited cell attachment to laminin, although the peptide itself was inactive (Graf, et al, (1987) Cell 48:989-996). Additional peptides have been synthesized corresponding to sequences in domain III, and a nine amino acid peptide (CDPGYIGSR) was found to be directly active in cell attachment of human fibrosarcoma HT-1080 and Chinese hamster ovary cells, and in binding to the 67 kD adhesion receptor. This peptide exhibits only 0.5-1% of the activity of laminin in cell attachment on a molar basis suggesting that it may represent only part of the structural determinants necessary for efficient laminin receptor binding. The peptide has also been found to promote the migration of B16F10 melanoma cells.
Preliminary studies suggest that a five amino acid peptide (5-mer), YIGSR, and a six amino acid peptide (6-mer), GYIGSR, contain sufficient information to allow cell adhesion, receptor binding, and cell migration activities (Graf, et al, (1987) Biochem 26:6896-6900; Iwamoto, et al, (1988) J Cell Physiol 134:287-291). The amide form of the pentapeptide has almost twice the activity of YIGSR itself, yet both the 5-mer and the 6-mer appeared to have lower but significant activity as compared to CDPGYIGSR.